(a) Field of the Invention
The present invention relates to mammalian expression vectors including a nuclear matrix attachment region of interferon β gene, and more particularly to pPGM-1, pPGM-2, and pPGM-3.
(b) Description of the Related Art
Various expression systems including microorganisms, plants, yeasts, insect cells, mammalian cells, etc. have been used to express and obtain target proteins in large quantities to apply to medical and industrial uses. Microorganisms are the easiest systems to use, and microorganism expression systems suitable for various applications have been developed and are commonly used.
However, microorganism expression systems have some limitations. The most serious limitation is that since protein expression and modification mechanisms (glycosylation, phosphorylation, amidation) of microorganisms differ from those of mammalian cells, even if the same gene is expressed in a microorganism system, the structure or characteristics of expressed proteins is not completely identical to the original protein. Therefore, production of recombinant proteins using microorganism expression systems frequently expresses proteins that are inactivated because modification does not occur after synthesis, or that partially differ in modification or structure even if they are not significantly different in function. In addition, the recombinant protein production process using microorganism expression systems should be accompanied by additional contaminant removal due to contamination of microorganisms, contamination of microorganism endotoxin, etc.
Meanwhile, mammalian expression systems, although they are the most suitable systems for expressing mammalian proteins, have not been easily industrialized because recombinant protein expression efficiency is low and thus the unit cost of production is high, and the mammalian cell handling process is difficult. Presently used industrial mammalian cell lines include CHO (Chinese Hamster Ovary), BHK (Baby Hamster Kidney), myeloma, etc., and an expression vector including the gene of interest is transfected into the mammalian cell line to express aimed foreign proteins.
Mammalian cells maintain various protein modification mechanisms including glycosylation, and protein obtainment and purification processes are easier when proteins are secreted to a culture medium. Most mammalian cells require complex additives such as serum protein, etc. in the culture process, while CHO cells can be cultured in a medium without serum and protein, rendering it the most suitable host for expression of recombinant proteins. In addition, characteristics of CHO cells are well known due to their having been used in many studies, and they have advantages of a high growth rate and that mass suspension culture is possible.
Generally, in order to express a transgene in a mammalian cell, a vector having a selection marker and a transgene are simultaneously transfected. Transfected cells are cultured and selected in a selection medium. However, expression frequency thereof is very low. One of the reasons is that these transgenes should be integrated in chromosomes of a host cell in mammalian cells contrary to the microorganism system. Additionally, even if stable transfectants are selected, the expression amount is difficult to predict. This is because gene integration positions differ according to cells, and expression aspects differ according to integration positions. Therefore, the copy number of transgenes in mammalian cells and the gene expression amount do not have an explicit correlation therebetween (Grindley et al., 1987, Trends Genet. 3, 16-22; Kucherlapati et al., 1984, Crit. Rev. Biochem. 16, 349-381; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-400). Gene expression in mammalian cells is mostly repressed by a nucleic acid base near the integration position, and thus stably integrated transgenes would often be expressed in a very low level (Eissenberg et al., 1991, Trends Genet. 7., 335-340; Palmiter et al., 1986, Annu. Rev. Genet. 20, 465-499).
Usability of nucleic acid factors for protecting transgene expression from position effects has been reported in many systems. As the nucleic acid factors, an insulator factor and a nuclear matrix attachment region (MAR) or scaffold attachment region (SAR), etc. can be used. Although operation mechanisms thereof have not been clarified, when included in transgene constructs, they induce position independent gene expression and the expression amount is determined by the copy number of gene (McKnight, R. A. et al., 1992, Proc. Natl. Acad. US. 89, 6943-6947).
Kalos et al. have combined the MAR factor of the human apolipoprotein B gene with a minimal promoter transgene construct and induced gene expression in mammalian cells to increase expression of the transcript by about 200 times (Kalos et al., 1995, Mol. Cell. Biol. 15, 198-207). Similarly, it has been reported that the MAR factor of the chicken lysozyme A gene and the SAR factor of human interferon β, etc. confer position-independent transgene expression in vertebrates (Eissenberg et al., 1991, Trends Genet. 7, 335-340; Klehr et al., 1991, Biochemistry 30, 1264-1270). However, no attempt to apply the MAR/SAR factor to substantially increase protein production in CHO cell lines has been reported, nor has industrial profit been identified.